NKT cells are considered unconventional T cells. First, they are restricted by a nonclassical MHC class I molecule, CD1d, which presents glycolipids; second, their TCR repertoire is very limited. After stimulation by their TCR, NKT cells rapidly release large amounts of cytokines, such as IL-4 and IFN-γ. Little is known about NKT cells present in lymph nodes. In the present report we show that NKT cells are differently distributed in various lymph nodes and are, for instance, abundant in pancreatic and mesenteric lymph nodes of C57BL/6 mice and nonobese diabetic mice. The high frequency of NKT cells in splanchnic lymph nodes is not simply a consequence of inflammatory signals, as draining lymph nodes still contain low frequencies of NKT cells after IFA or CFA injections. NKT cells from splanchnic lymph nodes harbor a Vβ repertoire similar to that of splenic and liver NKT cells, in contrast to peripheral NKT cells that are not biased toward Vβ8 segments. Analysis of cytokine production by NKT cells from splanchnic lymph nodes reveals that they produce at least as much IL-4 as IFN-γ, in contrast to NKT cells from other organs (spleen, liver, and peripheral lymph nodes), which produce much more IFN-γ than IL-4. These specific features of NKT cells from splanchnic lymph nodes might explain their protective action against the development of pathogenic Th1 cells in type 1 diabetes.